CA1132904A - Process for the recovery of heparin - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE
Heparin has been available for a long time as a naturally-occurring anticoagulant, and is now becoming increas-ingly inportant. A number of methods for isolating heparin from animal tissues such as the lungs, liver and intestinal mucus of pigs, cattle and sheep are known. According to the present invention, heparin is recovered from intestinal brine formed by treatment, with aqueous sodium chloride solution or solid sodium chloride in the presence of water, of animal intestines from which intestinal mucus has been removed. The heparin recovered is almost completely free of byproducts and heparin of good quality is obtained.

Description

~13Z904 , .
~ ne invention relates to the recovery of heparin from ~nimal tissues.
Heparin has been available in medicine for a long time as a naturally-occurring anticoagulant. Even in modern medicine it is still indispensible and in~eed is used to an ever increasing extent.
In xecent years, a number of processes have been developed, aimed at isolating the active substance, heparin, in an increasingly favourable manner from heparin-containing animal tissue, such as the lungs, liver and, morQ recently, especially from the intestinal mucus (mucosa) of pigs, cattle and sheep (GB-PS 754 885, DE~PS 1 228 241, US-PS 3 058 884, DE-PS 1 253 868).
Owing to the fact that they deteriorate easily, such raw materials have the disadvantage of a limited stor-ability and, although this can be prolonged by freezing, ,, I
in addition to greater technical complexity this leads to considerable increase in costs. Both the operatives , ...
; and the residents in the adjoining neighbourhood are not infrequently faced with unpleasant problems on account of the nuisance caused by odours associated with the processing.
In DE-PS 1 253 868 a few of the customary methods of recovering heparin are su~marised. The animal tis-sue, e.g. in_estinal mucus, is brought in the initial stage into contact with a hot aqueous salt solution in order to dissolve the heparin out of the cells. The ~ proportion of heparin in the resulting medium is extremely - small.
We have now found that heparin is present in the brine pro-duced in abbatoirs and plants for processing intestinal mucus, This brine is formed when animal intestines, from ~ .

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113'~904 which the int~stinal muc~is has pr~viously ~c!en rl~lnoved, are s-teeped for preserVaiion and dehydration in ~sodlur~ chloride solution, usllally at 0~C to room temperature and up to a ` maximum of 30C. A 3 to 5.6 molar sodium chloride solution - may for example, be used~
The present invention provides a process for recovering .:~
heparin from animal tissue, which comprises isolating heparin from intestinal brine formed by treatment with aqueous sodium chloride solution or solid sodium chloride in the presence of water, of animal intestines from which intestinal mucus has been removed. Usually the intestinal brine is produced from pigs, cattle or sheep intestines.
The present invention also provides heparin which has been recovered by the above process. The heparin may be recovered, for example, in the form of sodium heparinate or other physiologically tolerable salt of heparin.
The actual method of isolation of the starting material used according to the invention, and optional subsequent purification, may be carried out by a process known per se.
In view of DE-PS 1,253,868, it is to be regarded as positively surprising that the brine produced when steeping animal intestines in sodium chloride solution at low temperature under gentle conditions contains relatively large amounts of heparin. It was also unexpected that this heparin is almost completely free from chemically allied by-products. Thus the brine, previously regarded only as a waste product polluting the environment, represents a new source of raw material for heparin, now in short supply owing to a scarcity of raw material. Owing to the absence of impurities, the work-up to form crude heparin can be carried out in an especially favourable manner, and the complicated isolation methods (see US-PS 3,451,996 and GB-PS 1,221,784), : ' " 113Z904 accordin~ to the prior art are therefore unnecessary Further, the quality of heparin obtained is very much better than that of heparin preparations available ; ` on the market. Crude heparin isolat~d by known work--up ~ methods from the intestinal brine used according to the . .
invention is already in such a state of purity that subsequent purification yields heparin having far above 200 USP units per mg. The heparin preparations hitherto available commercially generally have heparin qualities of 150 USP units per mg, or, in a few cases, even 155 USP units per mg. One USP unit per mg is the specific activity which is obtained from the U.S.P. (United States Pharmacopoeia) assay, which measures the inhibi- -tion of the formation of clots in preserved sheep's ~- p]asma. 1 USP unit corresponds approximately to 1.1 international units ~I.U.). USP prescribes, for example, that heparin preparations (from intestinal mucus) must contain at least 140 USP units. In spite of numerous improvements, the activity values of com-mercially available heparin preparations continue to be unsatisfactory. L. W. Kananagh and L. B. Jaques ~ tArzneimittelforschung (Drug Research) 24, ~o. 12, 1942 "! (1974)] have succeeded in isolating heparin having a maximum of 175 USP units per mg, but this has involved repeated crystallisation of heparin in the form of its barium salt and has only been performed on a laboratory ~cale.
Sodium chloride solutions having a sodium chloride content of from 0.5 M to saturation are especially suit-able for preserving and dehydrating animal intestines, ~ and are preferably used in the process of the invention.
; Especially preferred are those solutions of sodium _3_ ' .

~132904 chloride having from a 3-molar content of sodium chlor-ide up to a saturation molar amount. The saturation molar amount is the molar amount of dissolved sodium chloride contained in a concentrated aqueous solution at room temperature in an abbatoir. (A concentrated aqueous sodium chloride solution has a strength of sub-stantialiy 26.4 wt.%) A concentrated aqueous sodium chloride solution may also be produced, for example, by ~ sprinkling the dripping wet intestines with sodium chloride.
Normally, the animal intestines, from which the mucus has been removed, are treated with the cold sodium chloride solutions for periods ranging from 2 hours to 3 days.
Usually, however, the treatment of the animal intestines is complete after 3 to 24 hours. After the preserved and dehydrated animal intestines have be2n r~moved from the sodium chloride solution, an intestinal brine having an unexpectedly high content of heparin is obtained.
Through the use according to the invention of such intestinal brines as a raw material, heparin has also been succ~ssfully isolated on an industrial scale in a purity such that it is in the form of an almost colour-less substance. Crude heparins available up to the presen~ day from other raw materials have to be bleached by expensive and wasteful methods (US-PS 3 179 566).
~oreover, since intestinal brine is a storable almost odourless raw material that does not suffer any loss in quality during long storage, chemical and other preservation methods can be dispensed with.
The present invention also provides the use of , 30 intestinal brine that has been obtained by steeping animal intestines, from which the iniestinal mucus has ` ~ been removed, in aqueous sodium chloride solution as a source :. . . . .

13'~904 of heparin, and also to the isolation of heparln from such sodi~n chloride solutions.
; The following Examples illustrate the invention.
In each case, the intestinal br~ne used was obtained by steeping animal intestines, from which the mucus had been removed, in aqueous sodium chloride solution of 26.4%
:
concentration at approximately 10C.
~xample 1 600 litres of methanol were added at room temperature in the course of ~ hour, while stirring, to 600 litres of intestinal brine in an apparatus of 1500 litres capacity equipped with a stirrer. Stirring was continued for a further ~ hour, after which the mixture was allowed to stand for about one hour.
The turbid supernatant liquid was then decanted into a second vessel, leaving behind the~ precipitated salt cake. The suspension obtained was again decanted, the supernatant liquid was discarded and the precipitate isolated by centrifuging. After drying in vacuo, 1.44 kg of a product that has about 10 USP units per mg was obtained. Up to about 45 % of it still consisted of sodium chloride.
An aliquot of the crude heparin thus obtained, cor-responding to about 425 USP units, or 45.2 g, was extracted three times with 200 ml of 2 M sodium chloride solution each time, stirring being carried out for one hour at 60C in each case. The combined extracts were diluted with water until the solution contained about ; 0.9 mole/litr~ of chloride ions. 200 ml of an anion-exchanger (Lewatit CA 9249) in the chloride form we_e added and stirring was carried out for one hour. The .. _ . . . ....................... . . . _ . .
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, . . :, ~ ' . : ' 113~9(~4 mixture was then suction filtered and washed ~ith about 200 ml of a 0.9 M sodium chloride solution. The ion-exchal~ger was then stirred for five hours at 40C ~!ith

2 M sodium chloride solution, filtered orf ~Jith suction and ~.~ashed with 2 M sodium chloride solution. The ` eluate was combined with the washing solution and pre-- cipitated with 1.5 times the volume of methanGl. ~he precipitate was centrifuged, washed first with a~o~t 50 ml of water-methanol (1 + 1.5 parts by volu~e) and then with about 50 ml of methanol, and dried.
198 g of sodium heparinate having 195 USP units per mg were obtained.
15.5 g of a sodium heparinate obtained in the a~ve manner and having about 200 USP units per mg ~ere dis-solved in 200 ml of 2 M sodium chloride solution. ~he solution was filtered through a suction filter and the residue washed first with 30 ml of 2 M sodium chloride solution and then with about 250 ml of completely desalinated water. The combined solutions were brought to a c'nloride molarity of 0.9 using completely desalina-ted ~ater. ~ne purification using Lewatit CA 9249 des-cribed above was then repeated.
The combined elution and washing solution was pro-cessed further as above. After washing and drying the precipitate, 11.2 g of sodium heparinate, having an activity of 251 USP units per mg, were obtained.
Example 2 In a manner similar to that described in GB-PS
754 885, 3 litres of intestinal brine were acidified with acetic acid to a ~H of 3.1 to 3.2 (ab~ut 50 ml), while stirring. After a few hours the mixture was decanted and the resldue was centrifuged. T~.e ,, , :; :

, pxecipitate was discarded, and the combined solutions were neutxalised with sodium hydroxide solution. I
The same volume of methanol was then slowly added while stirring. After 45 minutes the suspension waY
decanted from the precipitated sodium chloride. This suspension was allowed to stand for several hours, the supernatant liquid was decanted and the residue was centrifuged. The precipitate was dried in vacuo.

3.43 g having 21 USP units per mg were obtained.
An aliquot of the crude heparin thus obtained, cor-responding to about 425 000 USP~units, or 20.2 g, was purified in a marner analogous to that of Example 1.
10.65 g of sodium heparinate, having an activity of 262 USP units per mg, were obtained.
Exam~le 3 3 litres of intestinal brine were diluted with 12 litres of water to a chloride molarity of about 0.85 M.
50 g of Xieselguhr and a solution of 6 g of benzethonium chloride (= Hyamine 1622, Rohm & Haas) in 100 ml of water were then added. Afier a few hours decantation was car-ried out and the residue was filtered off with suction.
After being washed with water, the still moist precipi-tate was extracted three times by the method of DE-PS
1 228 241 with 200 ml of a 2 M solution of sodium chlor-ide each time. The combined extracts were precipitated with 2 parts by volume of methanol. The isolated and dried precipitate weighed 465 mg and had an activity of 155 USP units per mg.
Example 4 30 litres of intestinal brine were diluted to a chloride molarity of about 0.85 M with 120 litres of .

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~132904 water (cf. DE-PS 1 156 9~8). The mixture was then acidified to a pTI of 3.2 with acetic acid (about 50 mi).
After several hours a precipitate had settled and was centriuged after decanting the clear supernatant liquid.
After drying, 111 g of crude heparin having 6.5 USP
units per mg were obtained.
~n aliquot of the crude heparin thus obtained, cor-- responding to about 42S 000 USP units, or 65.5 g, was purified according to the method described in Example 1.
After the first purification stage over ion-exchanger, 196 g of sodium heparinate having 198 USP units per mg . . . .
were obtained.
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- ~ 15 g of a sodium heparinate obtained in the manner described above)and having about 200 USP units per mg, were dissolved in 200 ml of a 2N solution of sodium chloride. The solution was filtered through a suction filter, and the residue was washed first with 30 ml of a 2~ solution of calcium chloride and then with about 250 ml of completely desalinated water. The combined solutions were ~hen brought to a chloride molarity of 0.9 with completely desalinated water. Stirring, as .
above, with or.e litre of Lewatit CA 92a9 was carried out. The elution and washing were carried out with 2N
~; calcium chloride solution instead of with sodium chlor-,~, .
ide solution.
Ihe combined elution and washing solution was fur-ther worked up as above. After washing and drying the precipitate, 10.7 g of calcium heparinate, having an activity of 260 USP units per mg, were obtained.
Examnle 5 10 litres of intestinal brine wexe diluted with 40 litre~ of water to a chloride molarity of about 0.85 M.

~i~Z904 The solution was purnped (at the ~ate of about 3 litres per hour) through a filter over a column of ion-~xchanger, having a diameter of 26 r~m and a length of 380 mm. A medium basic macroporous anion-exchange resin in the Cl -form (Lewatit CA 9249) was used. Other anion-exchan~e resins may be used, such as Dowex 1-X-1 ~i described in DE-PS 1 253 868 for example.
The resin was then removed from the column, washed twice with 500 ml of an 0.9 M solution of sodium chlor-l0.. ide, and eluted with 400 ml of a 2 M solution of sodium chloride for 5 hours at ~0C, whi.le stirring. This ; solution was precipi'ated with 1.5 parts by volume of methanol. The isolated and dried precipitate weighed 1.2 g and had 149 USP units per mg.
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Claims (9)

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:

1. A process for the production of heparin in which heparin is isolated from an intestine-brine solution obtained by treatment with aqueous sodium chloride solution or solid sodium chloride in the presence of water of animal intestines at a tem-perature from 0 to 30°C whereby heparis of improved purity and molecular weight is obtained.

2. A process as claimed in Claim 1, wherein the treat-ment with sodium chloride solution is carried out at substantially room temperature.

3. A process as claimed in Claim 1, wherein the treat-ment with sodium chloride solution is carried out at 10°C or below.

4. A process as claimed in claim 1, wherein the treat-ment with sodium chloride solution is carried out for a period of from 2 hours to 3 days.

5. A process as claimed in Claim 3, wherein the period of treatment is from 3 to 24 hours.

6. A process as claimed in Claim 1, wherein the sodium chloride solution used has a sodium chloride content of from 0.5M
to saturation.

7. A process as claimed in Claim 5, wherein the sodium chloride solution used is from 3-molar to saturation molar with respect to sodium chloride.

8. A process as claimed in Claim 1, 2 or 3, wherein crude heparin is isolated by a method known per se and subsequently purified.

9. A process as claimed in Claims 1, 2 or 3, wherein heparin is recovered in the form of a physiologically tolerable salt thereof.